Colon cancer metastasis inhibitor

ABSTRACT

The present invention provides an agent for inhibiting metastasis of colorectal cancer and a method for inhibiting metastasis of colorectal cancer, which inhibit the function of Asef (i.e., binding activity to the APC gene product or guanine nucleotide exchange factor activity) that binds to the gene product of the tumor suppressor gene APC that plays an important role in tumorigenesis and in developmental processes, and/or inhibit the expression of the Asef gene.

This application is a National Stage Application of PCT/JP2003/010449,filed Aug. 19, 2003 and claims priority from Japanese Patent ApplicationNo. 2002-382083, filed Nov. 24, 2002, which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention relates to a method for inhibiting metastasis ofcolorectal cancer and an agent for inhibiting metastasis of colorectalcancer, which are characterized by inhibiting the function of Asef(APC-stimulated guanine nucleotide exchange factor) and/or inhibitingthe expression of Asef. More specifically, the present invention relatesto an agent for inhibiting metastasis of colorectal cancer, an agent forinhibiting Asef, a pharmaceutical composition, an agent for preventingand/or treating colorectal cancer, a method for inhibiting metastasis ofcolorectal cancer, and a method for preventing and/or treatingcolorectal cancer, which are characterized by inhibiting the expressionof Asef, inhibiting the binding of Asef to the gene product of APC(Adenomatous Polyposis Coli), or inhibiting the guanine nucleotideexchange factor (hereunder, referred to in abbreviated form as “GEF”)activity of Asef.

BACKGROUND ART

Asef is a protein that was found by the present inventors as acolorectal tumor suppressor gene-associated protein M1, which hasalready been disclosed and for which a patent application has been filed(Patent Reference 1 and Non-patent Reference 1). The protein consists of619 amino acid residues, and contains the Db1 homology (DH) domain, thepleckstrin homology (PH) domain and the Src homology 3 (SH3) domain inits amino acid sequence.

In terms of function, it is known that Asef has GEF activity specificfor Rac. Rac belongs to the Rho family, which is one of the smallGTP-binding protein families. More specifically, Asef binds to Rac tostimulate a GDP/GTP exchange reaction which results in the activation ofRac, thereby acting on NFκB, c-jun, SRE and the like, which are locateddownstream of the Rac related-intracellular signal transduction. The Rhofamily proteins play key roles in the reorganization of the actingnetwork, thereby regulating cell migration and cell-cell adhesion.Therefore, there is a possibility that Asef induces cellularlamellipodia (lobopodium) or cell membrane ruffling and participates incell migration and cell-cell adhesion.

It has been revealed that the binding of Asef to the gene product of thetumor suppressor gene APC via the armadillo repeat domain of the geneproduct. The GEF activity of Asef is positively regulated by the APCgene product. Actually, the induction of Asef-mediated cell membraneruffling or lamellipodia formation by the APC gene product is observedin MDCK cells that are canine kidney-derived epithelial-like cells.Further, Asef accumulates at the tips of microtubules in motile cellssimilarly to the APC gene product. Therefore, Asef may hold the key tocontrol cell migration when cells migrate from the crypt to the villustip of the colon.

Meanwhile, the tumor suppressor gene APC (Non-patent Reference 2) hasbeen isolated as a responsible gene for familial adenomatous polyposis(FAP). Mutation of the gene is observed in approximately 70% to 80% ofsporadic colorectal cancers. The APC gene product (hereunder, referredto as “APC”) is a giant protein of approximately 300 kDa that comprises2,843 amino acid residues. APC contains an armadillo repeat domain inthe amino acid sequence thereof that participates in protein-proteininteraction. Most somatic APC mutations observed in colorectal tumorcells occur within its central region called the “mutation clusterregion (MCR)” and result in the generation of truncated APCs that lackthe binding sites for microtubules, EB 1 or hDLG, and at least some ofthe sites for β-catenin and Axin (Non-patent References 4, 5, 6, 7 and8). However, the region of APC responsible for binding to Asef, thearmadillo repeat domain, is retained in most mutant APCs (Non-patentReferences 6, 7 and 8). APC has a function to bind to β-catenin, onekind of oncogene product, to induce its degradation (Non-patentReferences 2, 3, 4, 5 and 6). β-catenin, which is a Wnt/Wingless signaltransduction factor, binds to the cytoplasmic domain of cadherin andplays a role in cell adhesion, while it plays important roles indevelopmental processes and in tumorigenesis (Non-patent References 9and 10).

The amino acid sequence of Asef and the nucleotide sequence of its genehave been deposited with GenBank under the accession number AB042199.Further, the amino acid sequence of APC and the nucleotide sequence ofits gene have been deposited with GenBank under the accession numberNM000038.

Documents referred to in this specification are listed hereunder:

-   Patent Reference 1: Japanese Patent Laid-Open No. 2001-057888.-   Non-patent Reference 1: Kawasaki, Y., et al., Science, 2000, Vol.    289, p. 1194-1197.-   Non-patent Reference 2: Kinzler, K. W., et al., Cell, 1996, Vol.    87, p. 159-170.-   Non-patent Reference 3: Fearnhead, et al., Human Molecular Genetics,    2001, Vol. 10, p. 721-733.-   Non-patent Reference 4: Bienz, M., et al., Cell, 2000, Vol. 103, p.    311-320.-   Non-patent Reference 5: Perifer, M., et al., Science, 2000, Vol.    287, p. 1606-1609.-   Non-patent Reference 6: Akiyama, T., Cytokine and Growth Factor    Reviews, 2000, Vol. 11, p. 273-282.-   Non-patent Reference 7: Miyoshi, Y., et al., Human Molecular    Genetics, 1992, Vol. 1, p. 229-233.-   Non-patent Reference 8: Nagawa, H., et al., Human Mutation, 1993,    Vol. 2, p. 425-434.-   Non-patent Reference 9: Cell, 1996, Vol. 86, p. 391-399.-   Non-patent Reference 10: Nature, 1996, Vol. 382, p. 638-642.-   Non-patent Reference 11: Wong, M. H., et al., Proceeding of national    academy of science USA┘ 1996, Vol. 93, p. 9588-9593.-   Non-patent Reference 12: Oshima, H., et al., Cancer Research, 1997,    Vol. 57, p. 1644-1649.-   Non-patent Reference 13: Paddison, P. J., et al., Genes and    Development, 2002, Vol. 16, p. 948-958.

DISCLOSURE OF THE INVENTION

It is known that Asef binds to the gene product of the tumor suppressorgene APC which plays important roles in tumorigenesis and indevelopmental processes as described in the foregoing. However, thefunction of Asef in cells and the relation of Asef with diseases havenot yet been clarified. To clarify the function of Asef and regulate thefunction thereof makes it possible to prevent and treat diseasesattributable to Asef.

The present inventors hypothesized based on the GEF activity of Asef andits intracellular localization that Asef may participate in cellmigration and cell-cell adhesion, and found that Asef promotes themotility of colorectal tumor cells in colorectal cancers, particularlyin colorectal cancers in which APC mutations are observed, andparticipates in the metastasis. By utilizing this finding, the presentinventors found that metastasis of colorectal cancer is inhibited byinhibiting the function of Asef and/or inhibiting the expression of theAsef gene, and thereby complete the present invention.

That is, one aspect of the present invention relates to an agent forinhibiting metastasis of colorectal cancer, wherein the agent inhibitsthe function of Asef and/or inhibits the expression of the Asef gene.

Another aspect of the present invention relates to an agent forinhibiting metastasis of colorectal cancer, wherein the agent inhibitsthe expression of the Asef gene.

A further aspect of the present invention relates to an agent forinhibiting metastasis of colorectal cancer, wherein the agent inhibitsthe binding of Asef to the gene product of APC.

A still further aspect of the present invention relates to an agent forinhibiting metastasis of colorectal cancer, wherein the agent inhibitsthe guanine nucleotide exchange factor activity of Asef.

A further aspect of the present invention relates to a method forinhibiting metastasis of colorectal cancer, wherein the method comprisesinhibiting the function of Asef and/or inhibits the expression of theAsef gene.

A further aspect of the present invention relates to a method forinhibiting metastasis of colorectal cancer, wherein the method comprisesinhibiting the expression of the Asef gene.

A still further aspect of the present invention relates to a method forinhibiting metastasis of colorectal cancer, wherein the method comprisesinhibiting the binding of Asef to the gene product of APC.

A further aspect of the present invention relates to a method forinhibiting metastasis of colorectal cancer, wherein the method comprisesinhibiting the guanine nucleotide exchange factor activity of Asef.

A further aspect of the present invention relates to an agent forinhibiting Asef, wherein the agent utilizes RNA interference for theexpression of the Asef gene.

A still further aspect of the present invention relates to an agent forinhibiting Asef, comprising an oligonucleotide that exhibits an RNAinterference effect on the expression of the Asef gene.

A further aspect of the present invention relates to an oligonucleotidehaving the nucleotide sequence set forth in SEQ ID NO: 1 in the sequencelisting.

A further aspect of the present invention relates to an oligonucleotidehaving the nucleotide sequence set forth in SEQ ID NO: 2 in the sequencelisting.

A still further aspect of the present invention relates to anoligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 3in the sequence listing.

A further aspect of the present invention relates to an oligonucleotidehaving the nucleotide sequence set forth in SEQ ID NO: 4 in the sequencelisting.

A further aspect of the present invention relates to the preceding agentfor inhibiting Asef, comprising an oligonucleotide having the nucleotidesequence set forth in SEQ ID NO: 1 or 3 in the sequence listing.

A still further aspect of the present invention relates to a method forinhibiting Asef, wherein the method utilizes RNA interference on theexpression of the Asef gene.

A further aspect of the present invention relates to a method forinhibiting Asef, wherein the method comprises utilizing anoligonucleotide exhibiting an RNA interference effect on the expressionof the Asef gene.

A further aspect of the present invention relates to the precedingmethod for inhibiting Asef, wherein the method comprises utilizing anoligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 1or 3 in the sequence listing.

A still further aspect of the present invention relates to an agent forinhibiting metastasis of colorectal cancer, comprising any one of thepreceding agents for inhibiting Asef.

A further aspect of the present invention relates to an agent forinhibiting metastasis of colorectal cancer, comprising anoligonucleotide having the nucleotide sequence set forth in any one ofSEQ ID NOS: 1 to 4 in the sequence listing.

A further aspect of the present invention relates to a method forinhibiting metastasis of colorectal cancer, wherein the method uses anyone of the preceding agents for inhibiting Asef.

A still further aspect of the present invention relates to a method forinhibiting metastasis of colorectal cancer, wherein the method uses anoligonucleotide having the nucleotide sequence set forth in any one ofSEQ ID NOS: 1 to 4 in the sequence listing.

A further aspect of the present invention relates to a pharmaceuticalcomposition, comprising any one of the preceding agents for inhibitingmetastasis of colorectal cancer, or any one of the agents for inhibitingAsef.

A further aspect of the present invention relates to an agent forpreventing and/or treating colorectal cancer, comprising any one of thepreceding agents for inhibiting metastasis of colorectal cancer, or anyone of the agents for inhibiting Asef.

A still further aspect of the present invention relates to a method forpreventing and/or treating colorectal cancer, wherein the method usesany one of the preceding agents for inhibiting metastasis of colorectalcancer, or any one of the agents for inhibiting Asef.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates decreased cell-cell adhesion of MDCK cells infectedwith adenoviruses that contains DNA encoding Asef. As shown in thevertical axis, cell-cell adhesion is represented by a numerical valueobtained by dividing the number of cell clumps (Np) by the total numberof cells (Nc). Cells were infected with adenoviruses containing a geneencoding full-length Asef (denoted by “Asef-full”), a gene encoding thearmadillo repeat domain of the APC (denoted by “APC-arm”), a geneencoding an Asef mutant that lacks the APC-binding region (denoted by“Asef-ΔAPC”), or a gene encoding an Asef mutant that lacks the DH domain(denoted by “Asef-ΔDH”), as shown in the figure. The results are shownas mean±standard deviation (SD) obtained over three independentexperiments.

FIG. 2 illustrates that enhanced MDCK cell motility by the expression ofthe Asef gene was further increased by co-expression with an APC mutantthat contains the armadillo repeat domain (APC-arm, APC-876 andAPC-1309), while the enhanced motility by expression of the Asef gene,as well as the inherent cell motility, was decreased by expression ofthe Asef-ΔDH gene or the Asef-ABR (APC-binding region of Asef) gene. Theresults are shown as the relative migration as compared to that of theparental cells. The term “Mock” in the figure refers to cellstransfected with an empty vector.

FIG. 3 a illustrates the binding of Asef to truncated APC mutants inSW480 cells. Analysis of binding was carried out by immunoprecipitationusing an anti-Asef antibody (Anti-Asef). In the figure, the symbol+indicates that the antibody used was pre-incubated with antigen beforeimmunoprecipitation.

FIG. 3 b illustrates that Asef-ABR (APC-binding region of Asef)inhibited the interaction of IVT-APC-arm with GST-Asef-full in vitro ina dose dependent manner. In the figure, “MW.” represents a molecularmarker.

FIG. 4 illustrates that the motility of colorectal tumor SW480 cells isenhanced by expression of the Asef gene or the gene encoding Asef thatlacks the APC-binding region (Asef-ΔAPC), while the motility of variouscolorectal tumor cells (SW480, DLD-1, HCT15, WiDr and HCT116) did notchange or decreased when the gene encoding Asef lacking the GEF domain(Asef-ΔDH) was expressed. The results are shown as the relativemigration as compared to that of each cell expressing the LacZ gene as acontrol.

FIG. 5 illustrates that the short hairpin RNAs, shRNA-Asef and shRNA-APCwhich inhibit the expression of the Asef gene and the APC generespectively, both decreased the motility of colorectal tumor cellshaving an APC mutation (SW480 and WiDr), while no effect was observed onthe motility of colorectal tumor cells having normal APC (HCT116 andLS180). As controls for comparison, the short hairpin RNAs,mut-shRNA-Asef and mut-shRNA-APC, which do not inhibit the expression ofAsef gene or APC gene, were used. The results are shown as the relativemigration as compared to that of each cell transfected withmut-shRNA-Asef.

DETAILED DESCRIPTION OF THE INVENTION

The present invention claims the benefit of priority from JapanesePatent Application No. 2002-382083, which is incorporated herein byreference in its entirety.

Technical and scientific terms used herein have the meanings as normallyunderstood by those skilled in the art, unless otherwise defined.Various methods that are well known to those skilled in the art arereferenced herein. These reference materials, such as publishedmaterials disclosing known methods cited herein, are incorporated hereinby reference in their entirety.

Embodiments of the present invention are explained in further detailbelow. However, the detailed description below is exemplary and for thepurpose of explanation only, and is not intended to limit the scope ofthe present invention.

In the present invention, it was found that Asef decreases cell-celladhesion of epithelium-derived cells and also noticeably promotes themotility thereof. Further, it was found that these functions areregulated by APC, and particularly, it was found that truncated APCmutants that are identified in the majority of colorectal tumor cellsactivate Asef constitutively. Therefore, it is believed that formationof a complex between mutated APC and Asef contributes to aberrantmotility of colorectal tumor cells. That is, it is concluded that thecomplex may be involved in the upward migration of intestinal epithelialcells, more specifically, in the migration from the crypt to the villustip. Indeed, it has been reported that forced expression of the APC geneinduces aberrant cell migration in the intestinal epithelium (Non-patentReference 11). It has been reported that early adenoma cells in APCknockout mice exhibit a proliferation rate similar to that of normalcrypt epithelial cells, but lack directed migration along thecrypt-villus axis (Non-patent Reference 12).

Aberrant migratory behavior due to Asef activation by truncated APCmutants may be thus significant for both adenoma formation and tumorprogression to invasive malignancy. In addition, Asef mutants that lackthe GEF domain do not decrease cell-cell adhesion or do not promote cellmotility, resulting in the conclusion that GEF activity is important forsuch a function of Asef.

In the present invention, it was revealed that the motility ofcolorectal tumor cells expressing mutant APCs can be inhibited by usinga dominant-negative mutant that inhibits the binding of Asef to mutantAPCs, for example, a mutant consisting of the APC-binding region (aminoacid sequence from the 73^(rd) to the 126^(th) amino acid residue) inthe amino acid sequence of Asef or a mutant that lacks the GEF domain ofAsef. Also revealed was that the motility of colorectal tumor cellsexpressing mutant APCs can similarly be inhibited by inhibiting theexpression of the Asef gene or the APC gene. Further, it was found in anin vivo study using severe combined immunodeficient mice (SCID mice)that the tumorigenicity, proliferative growth and, moreover, metastasisof human colorectal tumor cells expressing the aforementioneddominant-negative mutants are inhibited in comparison to those of thecells that do not express the mutants. Such an inhibition was observedsimilarly in a study using cells that were obtained by cloning afterexpression of the mutants as human colorectal tumor cells expressing thedominant-negative mutants, and also in a study (mixed-population method)using a mixed population that was obtained from cells transformed withthe labeled mutants by using a cell sorter to concentrate the cellsexpressing the mutants to a density of 90% or more employing the labelas an indicator.

The inhibition of the function of Asef thus makes it possible to inhibitthe motility of cells and, further, to inhibit tumorigenicity of cellsand proliferative growth and/or metastasis of tumor cells. Since theinhibition of cell motility can also be achieved by inhibiting theexpression of the Asef gene or the APC gene, it is possible to inhibittumorigenicity of cells and proliferative growth and/or metastasis oftumor cells by inhibiting the expression of each these genes.

Based on the above-described findings, the present invention provides anagent for inhibiting metastasis of colorectal cancer and a method forinhibiting metastasis of colorectal cancer, which are characterized byinhibiting the function of Asef. The agent for inhibiting metastasis ofcolorectal cancer and the method for inhibiting metastasis of colorectalcancer are characterized by inhibiting the function of Asef and/orinhibiting the expression of the Asef gene.

Inhibition of the expression of the Asef gene can be carried out, forexample, by applying an RNA interference effect on the expression of theAsef gene. RNA interference is a method for inhibiting the expression ofa gene by using RNA, as has been reported in recent years (Non-patentReference 13). More specifically, the expression of the Asef gene can beinhibited by using an oligonucleotide that exhibits an RNA interferenceeffect on the expression of the Asef gene. Examples of theoligonucleotide can include a cDNA having the nucleotide sequence setforth in SEQ ID NO: 1 in the sequence listing. The complementary RNA(SEQ ID NO: 3 in the sequence listing) of the cDNA can also be used.Inhibition of the expression of the Asef gene can be carried out bytransfecting a cell with a vector containing the cDNA or with thecomplementary RNA thereof. Transfection of a cell with the vector orwith the RNA can be conducted utilizing a known method such aslipofection. Accordingly, an agent for inhibiting Asef comprising theaforementioned oligonucleotide is also included in the scope of thepresent invention. The agent for inhibiting Asef may contain one kind ofoligonucleotide, or may contain two or more kinds of oligonucleotide.Further, inhibition of the Asef gene expression may also be carried outby using an antisense oligonucleotide against the Asef gene. Theaforementioned oligonucleotide exhibiting an RNA interference effect orthe aforementioned antisense oligonucleotide can be obtained fromoligonucleotides that are designed on the basis of the nucleotidesequence of the Asef gene, by selecting oligonucleotides thatspecifically inhibit the expression of Asef using an Asef geneexpression system.

Inhibition of the function of Asef can be carried out, for example, byinhibiting the binding of Asef to APC, or inhibiting the GEF activity ofAsef. The binding of Asef to APC, which is the target of inhibition, ispreferably the binding of Asef to normal APC, more preferably thebinding of Asef to an APC mutant, further preferably the binding of Asefto a truncated APC mutant, and still more preferably the binding of Asefto a truncated APC mutant that contains an armadillo repeat domain.Examples of a truncated APC mutant that contains an armadillo repeatdomain include a polypeptide consisting of the consecutive amino acidresidues from the 1^(st) (the N terminus) to the 876^(th) residue of theamino acid sequence of APC, or a polypeptide consisting of theconsecutive amino acid residues from the 1^(st) (the N terminus) to the1309^(th) residue of the amino acid sequence of APC. These polypeptideswere identified as truncated APC mutants in most colorectal cancers andfamilial adenomatous polyposis (FAP).

Inhibition of the binding of Asef to APC can be carried out using adominant-negative Asef mutant for the binding. For example, an Asefmutant that can bind to APC but does not exhibit GEF activity can beused as an agent for inhibiting the binding of Asef to APC. Such an Asefmutant can be obtained by designing mutants based on the amino acidsequence of Asef and examining their binding activity to APC accordingto a conventional method. More specifically, a mutant that lacks the GEFdomain of Asef can be exemplified. Alternatively, a polypeptideconsisting of the APC-binding region (amino acid sequence from the73^(rd) to the 126^(th) amino acid residue) in the amino acid sequenceof Asef is preferably used. A polypeptide that inhibits the binding ofAsef to APC that is selected from polypeptides that are designed basedon the amino acid sequence of this polypeptide, can also be used.Further, inhibition of the binding of Asef to APC can also be carriedout by inhibiting the expression of the APC gene. Inhibition of APC geneexpression can be conducted by using an oligonucleotide that exhibits anRNA interference effect on the expression of the APC gene. Examples ofthe oligonucleotide can include a cDNA having the nucleotide sequenceset forth in SEQ ID NO: 2 in the sequence listing. Further, thecomplementary RNA (SEQ ID NO: 4 in the sequence listing) of the cDNA canalso be used. Alternatively, inhibition of the APC gene expression canbe carried out by using an antisense oligonucleotide against the APCgene. The aforementioned oligonucleotide exhibiting an RNA interferenceeffect or the aforementioned antisense oligonucleotide can be obtainedfrom oligonucleotides that are designed on the basis of the nucleotidesequence of the APC gene, by selecting oligonucleotides thatspecifically inhibit the expression of APC using an APC gene expressionsystem.

Inhibition of the GEF activity of Asef can be carried out, for example,by using an inhibitor of GEF activity that can be identified using Asef.Further, a compound that inhibits the expression of the Asef gene or acompound that inhibits the binding of Asef to APC may be identifiedusing the Asef gene or using Asef and APC, and the thus-identifiedcompound may be used. An assay system for identifying the compound canbe constructed utilizing a known screening system.

Metastasis of colorectal cancer can be inhibited by using an agent forinhibiting Asef that contains the above-described substance thatinhibits the function and/or the expression of Asef as an activeingredient. That is, an agent for inhibiting metastasis of colorectalcancer comprising an agent for inhibiting Asef and a method forinhibiting metastasis of colorectal cancer comprising using theaforementioned agent for inhibiting Asef are also included in the scopeof the present invention. More specifically, an agent for inhibitingmetastasis of colorectal cancer comprising an oligonucleotide having anyone of the nucleotide sequences set forth in SEQ ID NOS: 1 to 4 in thesequence listing and a method for inhibiting metastasis of colorectalcancer comprising using at least one of these oligonucleotides can beexemplified.

Tumorigenicity and metastasis of colorectal cancer can be inhibited byapplying the agent for inhibiting metastasis of colorectal cancer or theagent for inhibiting Asef of the present invention. More specifically,the above described agent for inhibiting metastasis of colorectal canceror the agent for inhibiting Asef can be used in the prevention and/ortreatment of colorectal cancer and colorectal cancer metastasis. Fromthis viewpoint, an agent for preventing and/or treating colorectalcancer comprising an effective amount of the aforementioned agent forinhibiting metastasis of colorectal cancer or the agent for inhibitingAsef as an active ingredient are also included in the scope of thepresent invention. Further, a method for preventing and/or treatingcolorectal cancer comprising using the aforementioned agent forinhibiting metastasis of colorectal cancer or the agent for inhibitingAsef can be also provided.

A pharmaceutical composition that includes the aforementioned agent forinhibiting metastasis of colorectal cancer or the agent for inhibitingAsef can be thus provided according to the present invention.

Suitable dosage ranges of the pharmaceutical composition of the presentinvention can be can be determined according to the following:effectiveness of the ingredients contained therein; the route ofadministration; the properties of the prescription; the characteristicsof the symptoms of the subject; and the judgment of the physician incharge. In general, a suitable dosage may fall, for example, within arange of approximately 0.01 μg to 100 mg per 1 kg of the body weight ofthe subject, and preferably within a range of approximately 0.1 μg to 1mg per 1 kg. However, a dosage may be altered using conventionalexperiments for optimization of a dosage that are well known in the art.The aforementioned dosage can be divided for administration once toseveral times a day. Alternatively, periodic administration once everyfew days or few weeks can be employed.

When using an oligonucleotide that is capable of inhibiting theexpression of the Asef gene or APC gene, it is possible to produce theoligonucleotide into the cell of the target by use of gene therapy. Thegene therapy can be performed by using a known method. For example, anon-viral transfection comprising administering the oligonucleotidedirectly by injection and a transfection using a virus vector can bothbe applied. A method is recommended for non-viral transfection thatcomprises administering a phospholipid vesicle such as a liposome thatcontains the oligonucleotide, as well as a method comprisingadministering the oligonucleotide directly by injection. A liposome foruse in this method can be more preferably exemplified by a cationicliposome. A vector used for a transfection using a virus vector, intowhich the oligonucleotide is incorporated, can be preferably exemplifiedby a DNA virus vector such as a retrovirus vector, an adenovirus vector,an adeno-associated virus vector and a vaccine virus vector, or a RNAvirus vector. Use of these virus vectors enables administration to becarried out effectively. Further, a method is recommended for atransfection using a virus vector that comprises administering aphospholipid vesicle such as a liposome that contains the vector.

A medicament of the present invention can be prepared as a medicamentthat contains only an active ingredient of the agent for inhibitingmetastasis of colorectal cancer or the agent for inhibiting Asef, but itis ordinarily prepared as a pharmaceutical composition using one or morekinds of a pharmaceutical carrier.

The amount of the active ingredient contained in the pharmaceuticalformulation of the present invention can be appropriately selected froma broad range. In general, a suitable amount may fall within a range ofapproximately 0.00001 to 70 wt %, preferably approximately 0.0001 to 5wt %.

Examples of the pharmaceutical carrier include a diluent or excipientsuch as a filler, expander, binder, wetting agent, disintegrator,surfactant, or lubricant that are normally used in accordance with theform of use of the formulation, and these can be suitably selected andused in accordance with the administration route of the formulation tobe obtained. Examples of the carrier include physiological saline,buffered physiological saline, dextrose, water, glycerol, ethanol, andmixtures thereof. A carrier is not limited to these examples, and anysubstance can be used according to one's desire as long as the substancecan be used for formulation of a common medicament.

The pharmaceutical composition of the present invention can be used as asolution formulation. It can also be used as a lyophilized formulationin order to preserve it, which can be used by dissolving it in water orin a buffered solution including physiological saline or the like toprepare it to a suitable concentration just before use.

When administering the pharmaceutical composition of the presentinvention, the pharmaceutical composition may be used alone or may beused together with other compounds or medicaments necessary for thetreatment.

In terms of a route of administration, it may be either systemicadministration or local administration. The route of administration thatis appropriate for a particular disease or symptomatic conditions shouldbe selected. As examples, parenteral administration including normalintravenous injection, intraarterial administration, subcutaneousadministration, intracutaneous administration and intramuscularadministration can be employed. Oral administration can be alsoemployed. Further, transmucosal administration or dermal administrationcan be employed. Direct administration to the neoplasm by an injectionor the like can be employed.

In terms of an administration form, various forms can be selected fromadministration forms that are known to those skilled in the art, andtypical examples thereof include an administration form of a solidformulation such as a tablet, pill, powder, powdered drug, fine granule,granule, or capsule, as well as an administration form of a liquidformulation such as an aqueous formulation, ethanol formulation,suspension, fat emulsion, liposome formulation, clathrate such ascyclodextrin, syrup, or an elixir. These can be further classifiedaccording to the administration route into an oral formulation,parenteral formulation (drip injection formulation or injectionformulation), nasal formulation, inhalant formulation, transvaginalformulation, suppositional formulation, sublingual agents, eye dropformulation, ear drop formulation, ointment formulation, creamformulation, transdermal absorption formulation, transmucosal absorptionformulation and the like, which can be respectively blended, formed andprepared according to conventional methods.

In general, when using the pharmaceutical composition of the presentinvention for gene therapy, the pharmaceutical composition is preferablyprepared as an injection formulation, drip injection formulation orliposome formulation. The pharmaceutical composition can also beprepared in a form that allows administration thereof together with asubstance that enhances the efficiency of gene transfer, such asprotamine.

Powders, pills, capsules, and tablets can be prepared using an excipientsuch as lactose, glucose, sucrose, or mannitol; a disintegrate agentsuch as starch or sodium alginate; a lubricant such as magnesiumstearate or talc; a binder such as polyvinyl alcohol, hydroxypropylcellulose, or gelatin; a surfactant such as fatty acid ester; aplasticizer such as glycerin, and the like. For preparation of a tabletor a capsule, a pharmaceutical carrier in a solid state is used.

A suspension can be prepared using water; sugars such as sucrose,sorbitol, or fructose; glycols such as PEG; and oils.

Injectable solutions can be prepared using a carrier comprising a saltsolution, a glucose solution or a mixture of salt water and a glucosesolution.

Inclusion into a liposome formulation can be conducted in the followingmanner: by dissolving the substance of interest in a solvent (e.g.,ethanol) to make a solution, adding a solution of phospholipidsdissolved in an organic solvent (e.g., chloroform), removing the solventby evaporation and adding a phosphate buffer thereto, agitating thesolution and then subjecting it to sonication followed by centrifugationto obtain a supernatant, and finally, filtrating the supernatant torecover liposomes.

A fat emulsion can be prepared in the following manner: by mixing thesubstance of interest, an oil ingredient (vegetable oil such as soybeanoil, sesame oil, olive oil, or MCT), an emulsifier (such as aphospholipid), and the like; heating the mixture to make a solution;adding water of a required quantity; and then emulsifying orhomogenizing by use of an emulsifier (a homogenizer, e.g., a highpressure jet type, an ultrasonic type, or the like). The fat emulsionmay be also lyophilized. For conducting lipid-emulsification, anauxiliary emulsifier may be added, and examples thereof include glycerinor saccharides (e.g., glucose, sorbitol, fructose, etc.).

Inclusion into a cyclodextrin formulation may be carried out in thefollowing manner: by dissolving the substance of interest in a solvent(e.g., ethanol); adding a solution of cyclodextrin dissolved in waterunder heating thereto; chilling the solution and filtering theprecipitates; and drying under sterilization. At this time, thecyclodextrin to be used may be appropriately selected from among thosehaving different void sizes (α, β, or γ type) in accordance with thebulkiness of the substance of interest.

EXAMPLES

Hereinafter, the present invention may be explained more particularlywith examples; however, the present invention is not limited to thefollowing examples.

First, the following definitions relate to Asef, APC, and the mutantsthereof that are used in the examples herein. The proteins and themutants are referred to in abbreviated form.

Asef-full is a protein consisting of the wild-type, full-length Asef. Itwas expressed as a haemagglutinin (HA)-tagged fusion protein (HA-taggedwild-type Asef) or a Glutathione S-transferase (GST) fusion protein(GST-Asef-full).

Asef-ΔAPC is a mutant that lacks the N-terminal APC-binding region ofAsef. This mutant possesses stronger GEF activity than wild-type Asef.

Asef-ΔDH is a mutant that lacks the DH domain (GEF domain) of Asef. Thismutant does not exhibit GEF activity.

Asef-ABR is a polypeptide consisting of the APC-binding region (aminoacid sequence from the 73^(rd) to the 126^(th) residue) in the aminoacid sequence of Asef. It was expressed as a maltose-binding protein(MBP) fusion protein (MBP-Asef-ABR).

APC-arm is a polypeptide consisting of the armadillo repeat domain ofAPC. It was expressed as a Myc-tagged fusion protein (Myc-taggedAPC-arm).

APC-876 is a polypeptide consisting of the consecutive amino acidresidues from the 1^(st) (the N terminus) to the 876^(th) residue of theamino acid sequence of APC, which contains the armadillo repeat domain.

APC-1309 is a polypeptide consisting of the consecutive amino acidresidues from the 1^(st) (the N terminus) to the 1309^(th) residue ofthe amino acid sequence of APC, which contains the armadillo repeatdomain.

APC-876 and APC-1309 are truncated APC mutants that were identified incolorectal tumors and familial adenomatous polyposis (FAP).

Adenoviruses that contain DNA encoding any of these proteins orpolypeptides were prepared by cloning polynucleotides that encode eachprotein into the pAdeno-X adenoviral vector using the Adeno-X™Expression System (Clontech, Palo Alto, Calif.). The term “AdAsef-full”hereunder refers to an adenovirus that contains DNA encoding Asef-full.Adenoviruses that contain DNA encoding the other proteins orpolypeptides described above are likewise represented hereunder byadding “Ad” to the designated name of each DNA.

Plasmids that contain DNA encoding any of the aforementioned proteins orpolypeptides were prepared according to a conventional method.

Cell culture and transfection of the aforementioned plasmids werecarried out as described in the following. MDCK cells (epithelial-likecell line established from a normal canine kidney) and WiDr cells werecultured in Dulbecco's modified Eagle's medium supplemented with 10%fetal calf serum (FCS). SW480 cells were cultured in Leibovitz's L-15medium supplemented with 10% FCS. DLD-1 cells and HCT15 cells werecultured in RPMI 1640 medium supplemented with 10% FCS. HCT116 cellswere cultured in McCoy's 5A medium supplemented with 10% FCS. Thesecells were transfected with the above-described plasmids usingLipofectAMINE 2000 (Life Technologies, Carlsbad, Calif.).

Preparation and expression of proteins were carried out in the followingmanner. Proteins fused to GST or MBP were synthesized in Escherichiacoli and isolated by absorption to glutathione Sepharose (GSH-Sepharose;Pharmacia, Buchinghamshire, UK) or amylose resin (New England Biolabs,Beverly, Mass.).

Short hairpin RNAs (hereunder, referred to in abbreviated form as“shRNA”), such as shRNA-Asef and shRNA-APC used in the RNA interferenceexperiments were designed to inhibit the expression of the Asef gene andthe APC gene, respectively. The nucleotide sequences of shRNA-Asef andshRNA-APC are set forth in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequencelisting, respectively. Further, mutations were added to shRNA-Asef andshRNA-APC to prepare shRNAs that did not inhibit the expression of theAsef gene and the APC gene. These are mut-shRNA-Asef and mut-shRNA-APC,which are set forth in SEQ ID NO: 5 and SEQ ID NO: 6 in the sequencelisting, respectively.

EXAMPLE 1

In order to examine the effects of Asef on cell-cell adhesion and cellmorphology, MDCK cells were infected with the above-describedadenoviruses. The adenoviruses used were AdAsef-full, AdAsef-ΔAPC,AdAsef-ΔDH and AdAPC-arm. AdLacZ was used as a control.Immunofluorescence staining showed that the infection efficiency of theadenoviruses to MDCK cells was 90% or more. Immunoblot analysis showedthat each of these viruses produces a protein of the expected size wheninfected into MDCK cells.

Cell morphology was examined as follows. Cells were planted in 12-welltissue culture plates to get 3.0×10⁴ cells per well. After 3 h ofincubation at 37° C., cells were infected with adenoviruses(multiplicity of infection (MOI) =200), cultured for a further 36 h andthen observed by a phase-contrast microscope. Cells infected withAdAsef-ΔAPC became flattened onto the substratum and exhibited membraneruffles and lamellipodia. In contrast, cells infected with AdAsef-ΔDHshowed no morphological changes and resembled uninfected cells.

Cell-cell adhesion was examined as follows. Infected cells were scrapedfrom plates in phosphate-buffered saline (PBS) containing 0.02%ethylenediamine tetraacetic acid (EDTA) and subjected to pipetting 20times. The number of cell clusters (particles) was then counted. Thecell-cell adhesion was evaluated from a value obtained by dividing thenumber of cell clusters by the total number of cells (Np/Nc). When cellswere dispersed by pipetting, cells infected with AdAsef-ΔAPC dissociatedefficiently, whereas uninfected cells and cells infected with AdAsef-ΔDHor AdLacZ remained as clusters (FIG. 1). These results showed that Asefhas a function to decrease cell-cell adhesion, and that its GEF activityis essential for this function.

Further, immunohistochemical analysis using an anti-E-cadherin antibodyshowed that overexpression of either AdAsef-full or AdAsef-ΔAPC resultedin decreased amounts of E-cadherin localized at the sites of cell-cellcontact and enhanced amounts of E-cadherin localized in the cytoplasm.Immunohistochemical analysis was conducted as follows. After 36 h ofadenovirus infection, MDCK cells were fixed with 3.7% formaldehyde inPBS. The fixed cells were double-stained with either a rat monoclonalantibody against E-cadherin (ECCD-2; Calbiochem, San Diego, Calif.) andtrimethylrhodamine isothiocyanate-conjugated phalloidin(TRITC-conjugated phalloidin: Molecular Probes, Eugene Oreg.), or therat monoclonal antibody against E-cadherin and a rabbit polyclonalantibody against β-catenin (SantaCruz Biotechnology, Santa Cruz, Calif.)for 60 min at room temperature. Staining patterns obtained withanti-E-cadherin antibody and anti-α-catenin antibody were visualizedwith fluorescein isothiocyanate-labelled anti-rat IgG antibody andTRITC-labelled anti-rabbit IgG antibody, respectively. The cells werephotographed with a Carl Zeiss LSM510 laser scanning microscope.Staining with anti-β-catenin antibody showed a decreased amount ofβ-catenin localized at the site of cell-cell contact, although thedecreased amount was not as prominent as that of E-cadherin. Incontrast, cells infected with AdAsef-ΔDH or AdLacZ did not show anychange in localization of E-cadherin or β-catenin. These results suggestthat GEF activity of Asef is important for the changes in thelocalization of these molecules. Immunoblot analysis of MDCK celllysates showed that the total amount of E-cadherin or β-catenin did notchange significantly upon infection with AdAsef-full or AdAsef-ΔAPC.These results demonstrated that the decrease in cell-cell adhesionresulting from the expression of the Asef gene is due to a decrease inE-cadherin and β-catenin at the sites of cell-cell contact.

EXAMPLE 2

The effects of Asef on cell motility were examined using MDCK cells thatwere made to express the Asef gene, the APC gene, or mutant genes ofthese using the plasmids described above. Cell motility was examined bycell migration assays using Transwell migration chambers. The chambersused for MDCK cells were 12 mm in diameter with a pore size of 12 μm(Costar Corporation, Cambridge Mass.). After 18 h of transfection,3.0×10⁴ cells of MDCK cells were added to the upper compartment of thechamber and allowed to migrate toward the underside of the upper chamberfor 18 h. Cell migration was determined by measuring the cells that hadmigrated to the lower side of the polycarbonate filters.

Cells infected with a plasmid that contains DNA encoding Asef-fullshowed enhanced motility as compared with parental cells (MDCK) orvector-transfected cells (Mock) (FIG. 2). Cells that were made toco-express the Asef-full gene together with any one of the APC-arm gene,the APC-876 gene and the APC-1309 gene were more motile than cellstransfected with the Asef-full gene alone. In the effect of APC on theability of Asef to promote cell-motility, APC-arm, APC-876 and APC-1309were stronger than APC-full. In contrast, APC-arm alone did not promotemigration. In addition, cells transfected with the Asef-ΔAPC gene showeda further enhanced migration reaction as compared with cellscotransfected with the Asef-full gene and the APC-arm gene. Theseresults showed that Asef has the potential to promote the migration ofMDCK cells. It was shown that this potential of Asef is further enhancedby APC, particularly a truncated APC mutant that contains an armadillorepeat domain (APC-Arm). In addition, Asef-ΔDH did not promote themigration of MDCK cells, indicating that the GEF activity of Asef isrequired for such migration stimulatory activity.

Meanwhile, when the Asef-ABR gene was expressed together with theAPC-1309 gene, enhancement of cell migration was almost completelyinhibited. Asef-ΔDH also inhibited APC-1309 mediated enhancement of cellmigration. These results indicate that the APC mutants, APC-879 andAPC-1309, which have been identified in colorectal cancers and FAP,interact with Asef, and enhance its activity, thereby promoting cellmigration. On the other hand, when the full-length APC gene wastransfected into MDCK cells, no enhancement of Asef-induced migrationwas observed (FIG. 2). This indicates that APC may not be an efficientactivator of Asef until APC is activated by truncation in colorectaltumor cells.

Next, the motility of SW480 cells that are known to include Asef andtruncated APC mutants was examined. When SW480 cells were transfectedwith plasmids that contain DNA encoding Asef-ABR, the migration of thecells decreased to about 50% of that of the parental cells or Mock cells(FIG. 2). Similarly, the migration of SW480 cells transfected withAsef-ΔDH plasmids decreased by to about 40%. These results demonstratedthat Asef-ABR or Asef-ΔDH expressed in cells acts on the binding of Asefto truncated APC mutants in a dominant-negative manner, therebyinhibiting the cell migration.

EXAMPLE 3

The binding of Asef to truncated APC mutants in colorectal tumor cellswas examined as follows. First, 5.0×10⁶ cells of SW480 cells were lysedin 500 μl of buffer A (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA,2 mM sodium vanadate (Na₃VO₄) and 10 mM sodium fluoride) containing 1%Triton X-100. The lysate was incubated with 2 μg of anti-Asef antibodyfor 1 h at 4° C., and then the immunocomplex was adsorbed to proteinG-Sepharose 6B for 2 h at 4° C. After washing extensively with buffer Acontaining 1% Triton X-100, the sample was resolved by SDS-PAGE, andtransferred to a polyvinylidene difluoride membrane filter (Immobilon P;Millipore, Bedford, Mass.). The blot was analyzed by immunoblot analysisusing alkaline phosphatase-conjugated mouse anti-rabbit IgG antibody(Promega, Madison, Wis.) as a second antibody. The rabbit anti-Asefpolyclonal antibody used was prepared by a conventional method(Non-patent Reference 1).

The results showed that Asef co-immunoprecipitated with the truncatedAPC mutant (FIG. 3 a). Co-immunoprecipitation of Asef and the APC mutantwas inhibited by preincubation of the antibody with an excess of theantigen for 2 h at 4° C. These results demonstrate that Asef isassociated with APC mutants in SW480 cells.

Next, co-immunoprecipitation of GST-Asef-full and APC-arm was conductedin vitro to examine the effect of Asef-ABR addition. First, APC-arm wasproduced by in vitro translation (IVT-APC-arm), and incubated withGST-Asef-full bound to Sepharose in the presence of MBP-Asef-ABR. Therelative amounts of APC-arm to MBP-Asef-ABR were varied as indicated inFIG. 3 b. APC-arm bound to GST-Asef-full-Sepharose was visualized bySDS-PAGE followed by autoradiography (top panel of FIG. 3 b).MBP-Asef-ABR added to the reaction mixture was visualized by subjectingthe gel to Coomassie blue staining (bottom panel of FIG. 3 b). Theresults showed that the amount of co-immunoprecipitate of APC-arm andGST-Asef-full decreased in a dose-dependent manner along with theincreasing amounts of Asef-ABR added. More specifically, it was revealedthat Asef-ABR inhibits the binding of Asef to the APC mutant in vitro.

The inhibition of the migration of SW480 cells was achieved usingAsef-ABR that inhibits the binding of Asef to the APC mutant in adominant negative manner.

EXAMPLE 4

Various colorectal tumor cell lines were infected with adenoviruses thatcontain DNA encoding Asef-full, Asef-ΔAPC or Asef-ΔDH, and assessed bycell migration assays in the same manner as in Example 2. The colorectaltumor cell lines used were SW480, DLD-1, HCT15, WiDr and HCT116. SW480cells, DLD-1 cells, HCT15 cells and WiDr cells contain truncated APCmutants. HCT116 cells contain normal APC but mutated β-catenin.

The results are shown in FIG. 4. When SW480 cells were infected withAdAsef-full or AdAsef-ΔAPC, their motility was enhanced. Further, whenSW480 cells, DLD-1 cells, HCT15 cells and WiDr cells were infected withAdAsef-ΔDH, their motility was partially inhibited. In contrast, themotility of HCT116 cells was not inhibited by AdAsef-ΔDH. This indicatesthat full-length APC in HCT116 is unable to induce the activation ofAsef. These results demonstrated that the activation of Asef is inducedin colorectal tumor cells that contain truncated APC mutants, while theactivation of Asef is not or is hardly induced in cells that containnormal APC. The results also showed that the activation is inhibited byAsef-ΔDH.

EXAMPLE 5

The interaction of Asef with APC mutants in the migration of colorectaltumor cells was investigated using RNA interference experiments. Theexperiments were carried out using the pSHAG-1 vector system (Non-patentReference 13). The colorectal tumor cell lines used were SW480 cells,WiDr cells, LS180 cells and HCT116 cells. SW480 cells and WiDr cellscontain truncated APC mutants. LS180 cells and HCT116 cells containnormal APC but mutated β-catenin.

Cell migration assays were carried out in the same manner as in Example2 to assess various colorectal tumor cells transfected with expressionvectors that contain either shRNA-Asef or shRNA-APC. The results showedthat SW480 cells and WiDr cells that were transfected with eithershRNA-Asef or shRNA-APC exhibited decreased motility as compared withcells that were transfected with mut-shRNA-Asef or mut-shRNA-APC (FIG.5). In contrast, this phenomenon was not observed in LS180 cells andHCT116 cells.

Next, immunoblot analysis was carried out in the same manner as inExample 3 to assess cells transfected with expression vectors thatcontain oligonucleotides encoding any one of shRNA-Asef, shRNA-APC,mut-shRNA-Asef and mut-shRNA-APC. Meanwhile, changes in α-tubulin weremeasured as a control. The results showed that shRNA-Asef and shRNA-APCalmost completely inhibited the expression of the Asef gene and the APCgene, respectively.

It was thus demonstrated that the motility of colorectal tumor cellsthat contain truncated APC mutants is decreased by inhibiting theexpression of the Asef gene or the APC gene. More specifically, theresults indicated that interaction of Asef with APC mutants plays animportant role in the migration of colorectal tumor cells.

EXAMPLE 6

Cells prepared by making the Asef dominant-negative mutant Asef-ABRexpress in human SW480 colorectal tumor cells were respectivelytransplanted to SCID mice to observe changes in their tumorigenicity orproliferation. Asef-ABR plasmids were transfected into SW480 colorectaltumor cells by lipofection. The thus-obtained 3 clones were respectivelycultured in vitro using L-15 medium containing G418 at a finalconcentration of 1 mg/ml, and then transplanted subcutaneously in theflanks of 8-week-old SCID mice at 1×10⁷ cells/0.1 ml/mouse (2 to 4individuals per group). Twenty days after transplanting the tumor cells,tumor lumps were excised to measure their weights. The weight of tumorlumps (T) of each mouse that was transplanted with the clone was dividedby the value of a control group (C) to get an inhibition ratio(abbreviated as “IR”) that was expressed as a percentage [IR(%)=T/C×100]. The expression of Asef-ABR in the transplanted cells wasconfirmed by a conventional method.

Decreased tumorigenicity or delayed proliferation was seen in 2 out of 3clones stably expressing Asef-ABR alone (Table 1). Thus, it is believedthat Asef participates in tumorigenicity or tumor cell proliferation.

TABLE 1 number of mice with tumor/ group weight ± SD (g) IR (%)transplanted with tumor SW480 0.496 ± 0.080 0 4/4 ABR-2 0.609 ± 0.069−22.7 3/3 ABR-8 0.000 ± 0.000 100 0/3 ABR-17 0.203 ± 0.056 59.1 4/4

EXAMPLE 7

Asef-ABR clones (see Example 6) that were prepared using the human HT29colorectal tumor cell line were transplanted into SCID mice to observechanges in their tumorigenicity or proliferation. 15 μg of Asef-ABRplasmids were transfected into 5×10⁶ cells of HT29 cells by lipofection.The thus-obtained 5 clones were respectively cultured in vitro usingDMEM medium containing G418 at a final concentration of 1 mg/ml, andthen transplanted into the spleen of 8-week-old SCID mice at 1×10⁶cells/0.05 ml/mouse (4 individuals per group). Eighteen days aftertransplanting the tumor cells, the mice were injected with ink via tailvein, and then sacrificed with bleeding under anesthesia with ether. Thespleen and the liver were excised to measure their weights.

Tumor formation in the spleen and the liver was not observed in 4 out of5 clones that are the stable transfectant expressing Asef-ABRdominant-negative mutant, which were prepared using HT29 cells (Table2). The same phenomenon was also observed in 3 clones prepared inExample 6 using SW480 colorectal tumor cells. Thus, it was demonstratedthat Asef participates in tumorigenicity and tumor cell proliferation.Further, no tumor formation in the liver was observed when using thestable transfectant expressing Asef-ABR dominant-negative mutant,indicating that Asef-ABR dominant-negative mutants inhibit tumormetastasis.

TABLE 2 group liver weight ± SD (g) spleen weight ± SD (mg) normal 1.340± 0.176 30.8 ± 7.1 parental cell line 2.236 ± 0.153 124.5 ± 20.4 (HT29)Asef-ABR-A7 1.584 ± 0.093 38.0 ± 3.9 Asef-ABR-B5 2.627 ± 0.392 129.3 ±13.5 Asef-ABR-C3 1.579 ± 0.124  44.3 ± 11.0 Asef-ABR-C12 1.526 ± 0.07037.8 ± 5.1 Asef-ABR-D11 1.359 ± 0.173 37.3 ± 4.6

INDUSTRIAL APPLICABILITY

In the present invention, it was found that Asef enhances the motilityof cells, and that it decreases cell-cell adhesion, and that thisfunction of Asef is activated by the gene product of the tumorsuppressor gene APC. Further, it was found that Asef enhances themotility of the colorectal tumor cells and participates in thetumorigenicity and metastasis thereof in colorectal cancers,particularly in colorectal cancers in which mutant APCs are observed.According to the present invention based on these findings, the agentfor inhibiting metastasis of colorectal cancer and the method forinhibiting metastasis of colorectal cancer, which inhibit the functionof Asef and/or inhibit the expression of Asef gene, were provided. Theseinventions have a significant effect for the prevention and/or treatmentof colorectal cancer and colorectal cancer metastasis.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1: Designed oligonucleotide based on the nucleotide sequenceof human Asef to inhibit the expression of the Asef gene.

-   SEQ ID NO: 2: Designed oligonucleotide based on the nucleotide    sequence of human APC to inhibit the expression of the APC gene.-   SEQ ID NO: 3: Designed oligonucleotide based on the nucleotide    sequence of human Asef to inhibit the expression of the Asef gene.-   SEQ ID NO: 4: Designed oligonucleotide based on the nucleotide    sequence of human APC to inhibit the expression of the APC gene.-   SEQ ID NO: 5: Designed oligonucleotide based on the nucleotide    sequence set forth in SEQ ID NO: 1.-   SEQ ID NO: 6: Designed oligonucleotide based on the nucleotide    sequence set forth in SEQ ID NO: 2.

1. An oligonucleotide consisting of the nucleotide sequence set forth inSEQ ID NO: 1 in the sequence listing.